U.S. patent application number 12/632639 was filed with the patent office on 2010-06-24 for method for high spatial resolution stochastic examination of a sample structure labeled with a substance.
This patent application is currently assigned to LEICA MICROSYSTEMS CMS GMBH. Invention is credited to Volker Seyfried, Jochen Sieber.
Application Number | 20100160613 12/632639 |
Document ID | / |
Family ID | 41716434 |
Filed Date | 2010-06-24 |
United States Patent
Application |
20100160613 |
Kind Code |
A1 |
Seyfried; Volker ; et
al. |
June 24, 2010 |
METHOD FOR HIGH SPATIAL RESOLUTION STOCHASTIC EXAMINATION OF A
SAMPLE STRUCTURE LABELED WITH A SUBSTANCE
Abstract
A method for high spatial resolution stochastic examination of a
biological sample structure labeled with a labeling substance is
described. The method comprises providing a biological sample
structure; choosing such a labeling substance that has molecules
present in a first state and in a second state, and the first and
second states differ from one another in at least one photophysical
property such that there is sufficient probability that one portion
of the molecules of the substance will be in the first state and
another portion of the molecules will be in the second state and
within which labeling substance a change of the state of the
molecules can occur spontaneously between the two states in both
directions; and labeling the biological sample structure with the
substance.
Inventors: |
Seyfried; Volker; (Nussloch,
DE) ; Sieber; Jochen; (Mannheim, DE) |
Correspondence
Address: |
ALEXANDER R SCHLEE;SCHLEE IP INTERNATIONAL P.C.
3770 HIGHLAND AVENUE, SUITE 203
MANHATTAN BEACH
CA
90266
US
|
Assignee: |
LEICA MICROSYSTEMS CMS GMBH
Wetzlar
DE
|
Family ID: |
41716434 |
Appl. No.: |
12/632639 |
Filed: |
December 7, 2009 |
Current U.S.
Class: |
530/402 |
Current CPC
Class: |
G01N 21/6428 20130101;
G01N 21/6458 20130101 |
Class at
Publication: |
530/402 |
International
Class: |
C07K 1/13 20060101
C07K001/13 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2008 |
DE |
DE 102008064164.2 |
Claims
1. A method for high spatial resolution stochastic examination of a
biological sample structure labeled with a labeling substance,
comprising: providing the biological sample structure; choosing
such a labeling substance that has molecules present in a first
state and in a second state, and the first and second states differ
from one another in at least one photophysical property such that
there is sufficient probability that one portion of the molecules
of the substance will be in the first state and another portion of
the molecules will be in the second state and within which labeling
substance a change of the state of the molecules can occur
spontaneously between the two states in both directions; and
labeling the biological sample structure with the substance.
2. The method according to claim 1, comprising choosing such a
labeling substance that has molecules present in at least one
additional state in addition to the first and second states,
wherein all states differ from one another in at least one
photophysical property such that there is sufficient probability
that a portion of the molecules will be in the first state, a
portion of the molecules will be in the second state, and a portion
of the molecules will be in the at least one additional state, it
being possible for a change to occur spontaneously between all
states in all directions.
3. The method according to claim 1, comprising choosing the
labeling substance such that at least one of the first and second
states is a non-fluorescent state, and at least one of the second
and first states is a fluorescent state.
4. The method according to claim 2, comprising choosing the
labeling substance such that at least one of the first, second and
additional states is a non-fluorescent state, and at least one of
the additional, second and first states is a fluorescent state.
5. The method according to claim 1, comprising choosing the
labeling substance such that the dwell time of the molecules in the
first or second state is a few microseconds.
6. The method according to claim 2, comprising choosing the
labeling substance such that the dwell time of the molecules in at
least one of the first and second and additional states is a few
microseconds.
7. The method according to claim 1, comprising choosing the
labeling substance such that a dwell time of the molecules in at
least one state is controllable by varying at least one external
parameter.
8. The method according to claim 1, comprising choosing the
labeling substance such that the rate of change between the states
is controllable by varying at least one external parameter.
9. The method according to claim 7, comprising choosing the
labeling substance such that the at least at least one external
parameter is at least one of the temperature, electric field, and
light.
10. The method according to claim 8, comprising choosing the
labeling substance such that the at least one external parameter is
at least one of the temperature, electric field, and light.
11. The method according to claim 7, comprising changing the
concentration of at least one of at least one component in the
biological sample structure and of at least one component in a
solution containing the biological sample structure.
12. The method according to claim 8, comprising changing the
concentration of at least one of at least one component in the
biological sample structure and of at least one component in a
solution containing the biological sample structure.
13. The method according to claim 7, comprising at least one of
replacing a solvent and changing a property of the solvent in a
solution containing the biological sample structure.
14. The method according to claim 1, comprising providing at least
one of a rate of change between the first and second states and the
dwell time in the first and second states dependent on the density
of the substance in the sample.
15. The method according to claim 1, comprising choosing the
labeling substance such that it includes at least two constituents
that can interact with each other in one of the first and second
state while in the other second or first state they cannot
interact.
16. The method according to claim 15, comprising choosing the
labeling substance such that a photophysical property in the first
or second state in which interaction is possible differs from the
photophysical property in the other second or first state where
interaction is not possible.
17. The method according to claim 16, comprising choosing the
labeling substance such that the constituents are the donor and the
acceptor of a FRET pair; and the desired signal is the fluorescence
of the acceptor.
18. The method according to claim 17, comprising choosing the
labeling substance such that the constituents are connected by a
linker; and at least one of the dwell time of the constituents in
one of the first and the second states and of the rate of change
between the states is controllable by at least one of the binding
of a component to the linker, an ion concentration in a solution
containing the biological sample structure, and the pH value.
19. The method according to claim 1, comprising choosing the
labeling substance or a substance complex as a protein or a protein
complex that can be present in two conformations or quaternary
structures which are present in a dynamic equilibrium and fluoresce
only in one of these states.
20. The method according to claim 1, comprising using the method
analogously to a method known as sptPALM (single particle tracking
PALM).
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the priority of the German patent
application DE 102008064164.2 having a filing date of Dec. 22,
2008. The entire content of this prior German patent application DE
102008064164.2 is herewith incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to a method for high spatial
resolution stochastic examination of a sample structure labeled
with a substance, whereby a biological structure is used as the
structure labeled with the substance, molecules of the substance
may be present in a first state and in a second state, and the
first and second states differ from one another in at least one
photophysical property.
[0003] Methods for high spatial resolution stochastic examination
of a sample structure labeled with a substance are known in the
art. In this regard, reference is made to WO 2006/127692 A2, US
2008/0032414 A1 and WO 2007/128434 A1, which describe stochastic
high-resolution and localization microscopy methods, which are
known as PALM, STORM, PALMIRA and GSDIM, respectively. Moreover, it
is known from the journal "Nature Methods", Vol. 5, No. 2, February
2008, pp. 155-157, that photoactivated localization microscopy
(PALM) can be performed on living cells, making it possible to
track movements of individual particles or molecules. This method
is also referred to as sptPALM (single particle tracking PALM).
[0004] In all of the known methods for high spatial resolution
stochastic examination of a sample structure labeled with a
substance, where a biological structure is used as the structure
labeled with the substance, molecules of the substance may be
present in a first state and in a second state. These first and
second states differ from one another in at least one photophysical
property. This photophysical property often consists in the ability
to fluoresce. In other words, there may be a first fluorescent
state and a second non-fluorescent state.
[0005] In the known methods, a substance with which a structure of
interest is labeled is actively switched between two states. In
PALM, PALMIRA or STORM, for example, the substance is switched from
a non-fluorescent state to a fluorescent state. This is usually
accomplished using light. In this process, care is taken to ensure
that only so many molecules of the substance are in the fluorescent
state, so that most of the signals that are detected by a
microscope having a CCD camera, for example, can be uniquely
associated with individual molecules. This is the fundamental idea
underlying these stochastic methods, in which a plurality of images
are recorded. After determining the centroid of the recorded
signals, a high-resolution image is constructed from the centroids
by superimposing the plurality of recorded images.
[0006] In the GSDIM method, for example, a fluorescent substance is
"pumped" into a dark state A, in which it does not fluoresce and
from which it may spontaneously return to the initial fluorescent
state B. Using a defined illumination, it can be achieved that only
a certain amount of molecules fluoresce simultaneously, and can
thus be localized. Analogously to the other high-resolution
stochastic methods, a high-resolution image can be generated by
recording a series of images of signals from single molecules and
determining and summing the individual centroids.
[0007] All of the previously known methods for high spatial
resolution stochastic examination of a sample structure labeled
with a substance require active switching between two states. This
switching is typically accomplished by light, for example by
illumination with a laser. The need for this active switching
operation makes the known methods complex.
SUMMARY OF THE INVENTION
[0008] It is, therefore, an object of the present invention to
provide a method of the above-mentioned type which allows a sample
structure labeled with a substance to be examined in a particularly
simple manner.
[0009] The above object is achieved in accordance with the present
invention by the method in question for high spatial resolution
stochastic examination of a sample structure labeled with a
substance that is further developed and refined by using a
substance within which there is sufficient probability that a large
enough portion of the molecules will be in the first state and a
large enough portion of the molecules will be in the second state,
and within which a change can occur spontaneously between the two
states in both directions.
DETAILED DESCRIPTION OF THE INVENTION
[0010] In accordance with the present invention, it was found that
the aforementioned object is achieved in a surprisingly simple
manner through suitable selection of the labeling substance.
Specifically, to this end, a substance is used in which a change
between two suitable states occurs spontaneously. There is no need
to activate the substance, or to specifically switch it from one
state to another using, for example, a laser. What is important in
the use of the substance according to the invention is that both
the first and second states be present with sufficient probability.
In other words, there is sufficient probability that a large enough
portion of the molecules will be intrinsically in the first state,
and that a large enough portion of the molecules will be
intrinsically in the second state. Ultimately, there is a dynamic
equilibrium between the two states, and a change may occur
spontaneously between the states.
[0011] Such spontaneous changing of states can be elegantly used in
stochastic high-resolution and localization microscopy because the
spontaneous changes between the states ensure that only a certain
number of molecules are in the particular state at a given time. It
never happens that all molecules are in the same state at the same
time. Thus, here too, as in corresponding methods known heretofore,
only a certain amount of molecules are in a state having the
desired photophysical property, which may, for example, be a
fluorescent state.
[0012] The method of the present invention may be referred to as
"spontaneous switching localization microscopy or SSLM", in which a
substance which spontaneously changes between states is used for
labeling the desired structure of a sample. This method is similar
to the conventional stochastic localization microscopy methods,
except that a substance as described above is used according to the
present invention.
[0013] The fundamental difference of the present invention from the
conventional methods is that the substance does not need to be
actively switched from one state to another. Rather, the changing
between the two states occurs intrinsically, so that no active
switching is needed.
[0014] Thus, the present invention provides a method for high
spatial resolution stochastic examination of a sample structure
labeled with a substance, whereby the labeled structure of the
sample can be examined in a particularly simple manner.
[0015] A typical method according to the present invention may be
implemented to include the following sequence of method steps:
Initially, a sample is labeled with the substance or with a tag
that binds the substance. Such methods for labeling a sample with a
tag that binds the substance are known in the art. In a subsequent
step, a series of images may be recorded of a sample. It is
important in this context that the amount of the substance that is
in a state in which it can emit detectable signals be only so large
that a substantial portion of the signals that come from single
molecules and are detected with the diffraction-limited resolution
of a microscope can still be uniquely associated with the emitting
single molecules. In a next step, the centroids of the single
molecules may be determined in the images of the series. Finally, a
high-resolution image may be constructed from the series of
centroid images.
[0016] In an advantageous embodiment of the method of the present
invention, the molecules of the substance may be present in at
least one additional state, all states differing from one another
in at least one photophysical property, and there being sufficient
probability that a large enough portion of the molecules will be in
the first state, a large enough portion of the molecules will be in
the second state, and a large enough portion of the molecules will
be in the least one additional state, it being possible for a
change to occur spontaneously between all states in all directions.
In other words, the method of the present invention is not limited
to a substance in which only two states exist. Rather, it is also
conceivable that three or more states may exist between which
changes may occur spontaneously to approximately the same or
different extent. A substance having a suitable number of states
may be suitably selected depending on the particular application.
Specifically, at least one state may be a non-fluorescent state,
and at least one other state may be a fluorescent state. However,
it would also be possible to use a different photophysical property
that distinguishes the respective states.
[0017] It is conceivable to use the method of the present invention
in applications where different average dwell times of the
molecules of the substance in one or in all of the states are
advantageous. Depending on the particular application, the dwell
time of the molecules in one or several states may be a few
microseconds, for example. Longer or shorter dwell times may also
be advantageous, depending on the particular application.
[0018] As a general principle, the dwell time of the molecules in
one or several states, or the rate of change between the states,
may be controllable by varying at least one external parameter.
Thus, the method may be adapted to the particular sample to be
examined.
[0019] One very simple approach provides that the at least external
parameter is the temperature, an electric field, or light. The
dwell time of the molecules in one or several states, or the rate
of change between the states, may be variable as a function of the
temperature level, the magnitude of the field, or the intensity of
the light. In this connection, consideration must be given to the
particular application.
[0020] Alternatively or additionally, control may be accomplished
by interaction with at least one component in the sample or with at
least one component in a solution containing the structure. To this
end, suitable components may be added into the sample or into the
solution as desired to achieve the desired control. Further
alternatively or additionally, control may be accomplished by
changing the pH value of a solution containing the structure.
[0021] Furthermore, it is advantageous if control is accomplished
by changing the concentration and/or a property of at least one
component in the sample or of at least one component in a solution
containing the structure. Moreover, control may be accomplished by
replacing a solvent or by changing a property of the solvent in a
solution containing the structure. Finally, control may be
accomplished by interaction with a local environment, preferably
with a surface or with a predeterminable state of a predeterminable
structure in the sample.
[0022] With regard to the aforementioned ways of achieving control,
it should be noted that the above listing is not meant to be
exhaustive. It is perfectly possible to implement other ways of
controlling the dwell time or the rate of change. In all cases,
with respect to the control options, consideration must be given to
the particular application. In principle, the rate of change
between the states, or the dwell time in the states, may also be
dependent on the density of the substance in the sample. Depending
on the particular application, a higher or lower density is to be
selected for the substance in the sample. Ultimately, depending on
the density of the substance in the sample, different rates of
change between the states may be advantageous, so that it is useful
to control the rate of change in a suitable manner.
[0023] In a specific embodiment, the substance may include at least
two constituents, and the constituents may interact with each other
in one state while in another state they may not. The two states
may exist in a dynamic equilibrium. Here too, the dwell time of the
constituents in the state with interaction and in the state without
interaction may be controllable as described above. Also, the rate
of change between the states may be controllable by varying at
least one external parameter as described above. In this case, too,
where the substance includes at least two constituents, a
photophysical property in the state in which interaction is
possible may differ from the photophysical property in the other
state.
[0024] Moreover, specifically, a desired signal may be obtained
from the substance only in the case where the constituents do not
interact with each other. Alternatively, depending on the
particular application, a desired signal may only be obtained from
the substance when the constituents do interact with each
other.
[0025] An example in which a signal is only obtained when the
constituents do not interact with each other may be what is known
as "quenching", i.e., reversible fluorescence quenching by a
quencher. An example in which a suitable signal is only obtained
when the constituents do interact with each other may be given in a
case where the constituents are the donor and the acceptor of a
FRET (fluorescence resonance energy transfer) pair, the desired
signal being the fluorescence of the acceptor.
[0026] The constituents may be connected by a linker, and the dwell
time of the constituents in one of the states, or the rate of
change between the states, may be controllable by the binding of a
component to the linker, by an ion concentration in a solution
containing the structure, or by the pH value. Furthermore, the
equilibrium between the two states may be controlled by the
temperature. Depending on the particular application, the
equilibrium may be advantageously shifted in such a way that either
the one or the other state is present in a larger proportion and
with greater probability.
[0027] For example, in the case of Chameleon, a Ca2+ sensor
composed of a FRET pair of two fluorescent proteins bound by a
linker, the conformation, and thus the FRET efficiency and the
intensity of the acceptor signal, may be controlled via the Ca2+
concentration.
[0028] In another specific embodiment, the substance or a substance
complex, may be present in two conformations or quaternary
structures which are present in a dynamic equilibrium and
fluoresce, for example, only in one of these states. The substance
or substance complex used may, for example, be a protein or a
protein complex. The equilibrium, i.e., the dwell time or the rate
of change, may be controlled in the manner described above.
[0029] In order to provide a particularly efficient examination
method, a plurality of sample structures labeled with different
substances may be simultaneously or sequentially examined by the
same detector or by different detectors and be differentiated on
the basis of different properties of the respective detected
signals so as to obtain a high-resolution image of a plurality of
structures. The different properties may include, for example,
different emission and excitation spectra.
[0030] Efficient and specific illumination of the sample may be
accomplished using SPIM (Selective Plane Illumination Microscopy)
illumination, TIRF (Total Internal Reflection Fluorescence)
illumination, or wide-field illumination. In addition to performing
the detection using one camera, it may be advantageous to perform
the detection using a plurality of cameras which also allow 3D
information to be obtained in high-resolution. With regard to the
analysis of the data obtained, it is possible to use conventional
analysis methods, especially those allowing 3D representations to
be obtained via the shape and magnitude of the detected
signals.
[0031] Further advantageously, the method of the present invention
may be used in a method analogous to the one known as sptPALM
(single particle tracking PALM). In this case, in place of
generating a high-resolution image from the series of centroid
images, it is possible to analyze movements of the respective
single molecules between the images. Analogously to sptPALM, the
advantage over conventional single particle tracking methods is the
ability to always read out different molecules that are currently
in the state in which they emit the detectable signal. Thus, it is
possible, for example, to obtain a much better statistic for a cell
than would be possible using conventional single particle tracking
methods. The method of the present invention has the advantage that
no active switching between the states is needed.
[0032] The teaching of the present invention may be advantageously
embodied and refined in various ways. In this regard, reference is
made, on the one hand, to the claims that are subordinate to claim
1 and, on the other hand, to the following description of preferred
exemplary embodiments of the invention which makes reference to the
drawing. In conjunction with the explanation of the preferred
exemplary embodiments of the present invention with reference to
the drawing, an explanation is also given of generally preferred
embodiments and refinements of the teaching.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 is a schematic view of an exemplary embodiment of a
substance that can be used in the method of the present invention,
said substance including two constituents connected by a linker;
and
[0034] FIG. 2 is a schematic view of another exemplary embodiment
of a suitable substance, said substance including three
constituents connected by linkers.
DETAILED DESCRIPTION OF THE DRAWINGS
[0035] In a schematic representation, FIG. 1 shows a substance that
can be used in an exemplary embodiment of the method of the present
invention, said substance including two constituents 1 and 2 which
are connected to each other by a flexible linker 3. The substance
can be present in two states A and B, which exist in a dynamic
equilibrium. Constituents 1 and 2 can interact with each other in
one of these states while in the other they cannot.
[0036] In this connection, a signal may only be obtained when the
constituents do not interact with each other or when they do
interact with each other. The states A and B exist with
controllable probabilities, it being possible for a change to occur
spontaneously between the two states in both directions.
[0037] In a schematic representation, FIG. 2 shows another
substance which can be used in the method of the present invention.
The substance shown in FIG. 2 includes three constituents 1, 2 and
4, constituents 1 and 2 being connected to each other by a linker 3
and constituents 2 and 4 being connected to each other by a linker
5.
[0038] FIG. 2 shows intermediate states between the states A and B.
These intermediate states can also occur spontaneously. In the case
of the intermediate state shown in the upper portion of FIG. 2,
constituents 1 and 2 are adjacent or bound to each other, while in
the case of the intermediate state shown in the lower portion of
FIG. 2, constituents 2 and 4 are adjacent or bound to each other.
In state A, none of the constituents 1, 2 or 4 are adjacent to each
other, whereas in state B, all of the constituents 1, 2 and 4 are
adjacent to each other or bound, respectively.
[0039] In the case of the exemplary embodiment of a substance
illustrated in FIG. 2, the dwell time of constituents 1, 2 and 4 in
states A and B, or the rate of change between states A and B, is
also controllable.
[0040] Finally, it should be emphasized that the exemplary
embodiments discussed above are merely intended to illustrate the
claimed teaching, but not to limit it to such embodiments.
LIST OF REFERENCE NUMERALS
[0041] 1 constituent [0042] 2 constituent [0043] 3 linker [0044] 4
constituent [0045] 5 linker [0046] A state [0047] B state
* * * * *